The present invention relates to novel antibodies that are selective for the IGF-1R cell surface receptor. Also included is derivation of recombinant antibodies, e.g., chimeric, humanized or veneered versions including single chain Fv fragments (scFv) from the mammalian antibodies detailed herein and designated “12B1”. The invention likewise comprises utilization of the murine or recombinant antibodies derived therefrom in detecting and diagnosing pathological hyperproliferative oncogenic disorders associated with expression of IGF-1R. In certain embodiments, the disorders are oncogenic disorders associated with increased expression of IGF-1R polypeptide relative to normal or any other pathology connected with the overexpression of IGF-1R. Use of the recombinant antibodies as a prognostic marker and kits for diagnosis of illnesses connected with the overexpression of the IGF-IR receptor are also disclosed. The amino acid and nucleic acid sequences coding for these antibodies as well as methods of assessing the therapeutic efficacy of a treatment regiment comprising an IGF-1R specific modulating moiety is also disclosed.
Various growth factors, including insulin-like growth factors (IGF), e.g., insulin-like growth factor-I and insulin-like growth factor-II have been implicated in exerting mitogenic activity on various cell types such as tumor cells. IGFs are structurally similar to insulin, and have been implicated as a therapeutic tool in a variety of diseases and injuries. Insulin-like growth factor-I (IGF-I) is a 7649-dalton polypeptide with a pI of 8.4 that circulates in plasma in high concentrations and is detectable in most tissues (Rinderknecht and Humbel, Proc. Natl. Acad. Sci. USA, 73: 2365 (1976); Rinderknecht and Humbel, J. Biol. Chem., 253: 2769 (1978)). IGF-I stimulates cell differentiation and cell proliferation, and is required by most mammalian cell types for sustained proliferation. These cell types include, among others, human diploid fibroblasts, epithelial cells, smooth muscle cells, T lymphocytes, neural cells, myeloid cells, chondrocytes, osteoblasts and bone marrow stem cells. Each of these growth factors exerts its mitogenic effects by binding to a common receptor named the insulin-like growth factor receptor-1 (IGF1R) (Sepp-Lorenzino, (1998) Breast Cancer Research and Treatment 47:235). See also Klapper, et al., (1983) Endocrinol. 112:2215 and Rinderknecht, et al., (1978) Febs. Lett. 89:283. There is a large body of literature on the actions and activities of IGFs (IGF-1, IGF-2, and IGF variants). See Van Wyk et al., Recent Prog. Horm. Res., 30: 259 (1974); Binoux, Ann. Endocrinol., 41: 157 (1980); Clemmons and Van Wyk, Handbook Exp. Pharmacol., 57: 161 (1981); Baxter, Adv. Clin. Chem., 25:49 (1986); U.S. Pat. No. 4,988,675; WO 91/03253; WO 93/23071).
The IGF system is also composed of membrane-bound receptors for IGF-1, IGF-2, and insulin. The Type 1 IGF receptor (IGF-1R) is closely related to the insulin receptor (IR) in structure and shares some of its signaling pathways (Jones and Clemmons, Endocr. Rev., 16: 3-34 (1995); Ullrich et al., Cell 61: 203 212, 1990), and is structurally similar to the insulin receptor (Ullrich et al., EMBO J. 5: 2503 2512, 1986)). Since IGF-1 and IGF-2 bind to IGF-1R with a much higher affinity than to the insulin receptor, it is most likely that most of the effects of IGF-1 and IGF-2 are mediated by IGF-1R (Humbel, Eur. J. Biochem. 190:445-462 (1990); Ballard et al., “Does IGF-I ever act through the insulin receptor?”, in Baxter et al. (Eds.), The Insulin-Like Growth Factors and Their Regulatory Proteins, (Amsterdam: Elsevier, 1994), pp. 131-138). The crystal structure of the first three domains of IGF-1R has been determined (Garrett et al., Nature, 394, 395-399 (1998)). While similar in structure, IGF-1R and IR serve different physiological functions in that IR is primarily involved in metabolic functions whereas IGF-1R mediates growth and differentiation. For a review of the wide variety of cell types for which IGF-I/IGF-I receptor interaction mediates cell proliferation, see Goldring et al., Eukar. Gene Express., 1:31 326 (1991). The IGF-2 receptor, on the other hand, is a clearance receptor that appears not to transmit an intracellular signal (Jones and Clemmons, supra).
The insulin-like growth factor I receptor (IGF-1R) is a glycoprotein of molecular weight approximately 350,000. It is a hetero-tetrameric receptor of which each half-linked by disulfide bridges—is composed of an extracellular α-subunit and of a transmembrane β-subunit. The IGF-I receptor is composed of two types of subunits: an alpha subunit (a 130 135 kD protein that is entirely extracellular and functions in ligand binding) and a beta subunit (a 95-kD transmembrane protein, with transmembrane and cytoplasmic domains). The IGF-IR is initially synthesized as a single chain proreceptor polypeptide which is processed by glycosylation, proteolytic cleavage, and covalent bonding to assemble into a mature 460-kD heterotetramer comprising two alpha-subunits and two beta-subunits. The beta subunit(s) possesses ligand-activated tyrosine kinase activity. This activity is implicated in the signaling pathways mediating ligand action which involve autophosphorylation of the beta-subunit and phosphorylation of IGF-IR substrates.
IGF-IR binds IGF I and IGF II with nanomolar affinity, e.g., Kd of 1×10−9 nM but is capable of binding to insulin with an affinity 100 to 1000 times less. Representative nanomolar affinity values may be found in FEBS Letters, vol. 565, pages 19-22 (2004), the entire content of which is incorporated by reference herein. Conversely, the IR binds insulin with a very high affinity although the IGFs only bind to the insulin receptor with a 100 times lower affinity. The tyrosine kinase domain of IGF-IR and of IR has a very high sequence homology although the zones of weaker homology respectively concern the cysteine-rich region situated on the α-subunit and the C-terminal part of the β-subunit. The sequence differences observed in the α-subunit are situated in the binding zone of the ligands and are therefore at the origin of the relative affinities of IGF-IR and of IR for the IGFs and insulin respectively. The differences in the C-terminal part of the β-subunit result in a divergence in the signalling pathways of the two receptors; IGF-IR mediating mitogenic, differentiation and antiapoptosis effects, while the activation of the IR principally involves effects at the level of the metabolic pathways (Baserga et al., Biochim. Biophys. Acta, 1332: F105-126, 1997; Baserga R., Exp. Cell. Res., 253:1-6, 1999).
The first step in the transduction pathway leading to IGF-1-stimulated cellular proliferation or differentiation is binding of IGF-I or IGF-II (or insulin) at physiological concentrations to the IGF-I receptor. Interaction of IGFs with IGF1R activates the receptor by triggering autophosphorylation of the receptor on tyrosine residues (Butler, et al., (1998) Comparative Biochemistry and Physiology 121:19). Once activated, IGF1R, in turn, phosphorylates intracellular targets to activate cellular signaling pathways. This receptor activation is critical for stimulation of tumor cell growth and survival. Therefore, inhibition of IGF1R activity represents a valuable potential method to treat or prevent growth of human cancers and other proliferative diseases.
There is considerable evidence for a role for IGF-I and/or IGF-IR in the maintenance of tumor cells in vitro and in vivo. For example, individuals with “high normal” levels of IGF-I have an increased risk of common cancers compared to individuals with IGF-I levels in the “low normal” range (Rosen et al., Trends Endocrinol. Metab. 10: 136 41, 1999). For a review of the role IGF-I/IGF-I receptor interaction plays in the growth of a variety of human tumors, see Macaulay, Br. J. Cancer, 65: 311 320, 1992. In addition to playing a key role in normal cell growth and development, IGF-1R signaling has also been implicated as playing a critical role in growth of tumor cells, cell transformation, and tumorigenesis. See Baserga, Cancer Res., 55:249-252 (1995); for a review, see Khandwala et al., Endocr. Rev. 21: 215-244 (2000)); Daughaday and Rotwein, Endocrine Rev., 10:68-91 (1989). Recent data impel the conclusion that IGF-IR is expressed in a great variety of tumors and of tumor lines and the IGFs amplify the tumor growth via their attachment to IGF-IR. Indeed, the crucial discovery which has clearly demonstrated the major role played by IGF-IR in the transformation has been the demonstration that the R-cells, in which the gene coding for IGF-IR has been inactivated, are totally refractory to transformation by different agents which are usually capable of transforming the cells, such as the E5 protein of bovine papilloma virus, an overexpression of EGFR or of PDGFR, the T antigen of SV 40, activated ras or the combination of these two last factors (Sell C. et al., Proc. Natl. Acad. Sci., USA, 90: 11217-11221, 1993; Sell C. et al., Mol. Cell. Biol., 14:3604-3612, 1994; Morrione A. J., Virol., 69:5300-5303, 1995; Coppola D. et al., Mol. Cell. Biol., 14:4588-4595, 1994; DeAngelis T et al., J. Cell. Physiol., 164:214-221, 1995). Other key examples supporting this hypothesis include loss of metastatic phenotype of murine carcinoma cells by treatment with antisense RNA to the IGF-1R (Long et al., Cancer Res., 55:1006-1009 (1995)) and the in vitro inhibition of human melanoma cell motility (Stracke et al., J. Biol. Chem., 264:21554-21559 (1989)) and of human breast cancer cell growth by the addition of IGF-1R antibodies (Rohlik et al., Biochem. Biophys. Res. Commun., 149:276-281 (1987)).
Other arguments in favor of the role of IGF-IR in carcinogenesis come from studies using murine monoclonal antibodies directed against the receptor or using negative dominants of IGF-IR. In effect, murine monoclonal antibodies directed against IGF-IR inhibit the proliferation of numerous cell lines in culture and the growth of tumor cells in vivo (Arteaga C. et al., Cancer Res., 49:6237-6241, 1989; Li et al., Biochem. Biophys. Res. Com., 196:92-98, 1993; Zia F et al., J. Cell. Biol., 24:269-275, 1996; Scotlandi K et al., Cancer Res., 58:4127-4131, 1998). It has likewise been shown in the works of Jiang et al. (Oncogene, 18:6071-6077, 1999) that a negative dominant of IGF-IR is capable of inhibiting tumor proliferation.
Using antisense expression vectors or antisense oligonucleotides to the IGF-IR RNA, it has been shown that interference with IGF-IR leads to inhibition of IGF-I-mediated or IGF-II-mediated cell growth (see, e.g., Wraight et al., Nat. Biotech. 18: 521 526, 2000). The antisense strategy was successful in inhibiting cellular proliferation in several normal cell types and in human tumor cell lines. Growth has also been inhibited using peptide analogues of IGF-I (Pietrzkowski et al., Cell Growth & Diff. 3: 199 205, 1992; and Pietrzkowski et al., Mol. Cell. Biol., 12: 3883 3889, 1992), or a vector expressing an antisense RNA to the IGF-I RNA (Trojan et al., Science 259: 94 97, 1992.
IGF-IR levels are elevated in tumors of lung (Kaiser et al., J. Cancer Res. Clin. Oncol. 119: 665 668, 1993; Moody et al., Life Sciences 52: 1161 1173, 1993; Macauley et al., Cancer Res., 50: 2511 2517, 1990), breast (Pollak et al., Cancer Lett. 38: 223 230, 1987; Foekens et al., Cancer Res. 49: 7002 7009, 1989; Cullen et al., Cancer Res. 49: 7002 7009, 1990; Arteaga et al., J. Clin. Invest. 84: 1418 1423, 1989), prostate and colon (Remaole-Bennet et al., J. Clin. Endocrinol. Metab. 75: 609 616, 1992; Guo et al., Gastroenterol. 102: 1101 1108, 1992).
Elevated serum levels of IGF-1 have been shown to be associated with increased risks of prostate cancer, and may be an earlier predictor of onset than prostate-specific antigen (PSA; J. M. Chan et al., 1998, Science 279:563-566).
There also appears to be a relationship between high levels of IGF-1 and/or IGF-1R and breast cancer (L. C. Happerfield et al., 1997, J. Pathol. 183:412-417). Breast cancers express IGF-2 and IGF-1R, providing all the required effectors for an autocrine-loop-based proliferation paradigm (Quinn et al., J. Biol. Chem., 271:11477-11483 (1996); Steller et al., Cancer Res., 56:1761-1765 (1996)). Indeed, IGF-1R is overexpressed in 40% of all breast cancer cell lines (Pandini, et al., (1999) Cancer Res. 5:1935) and in 15% of lung cancer cell lines. In breast cancer tumor tissue, IGF1R is overexpressed 6-14 fold and IGF1R exhibits 2-4 fold higher kinase activity as compared to normal tissue (Webster, et al., (1996) Cancer Res. 56:2781 and Pekonen, et al., (1998) Cancer Res. 48:1343). In fact, a positive correlation was observed between circulating IGF-1 and breast cancer among pre-menopausal women (S. E. Hankinson et al., 1998, Lancet 351:1393-1396). A poor prognosis for breast cancer patients was correlated to the expression of IGF-1R positive and estrogen receptor (ER) negative cells (A. A. Butler et al., 1998, Cancer Res. 58:3021-3027). Recently, investigators have identified hybrid IGF-1R/IR receptors found in several breast cancer cell lines (G. Pandini et al., 1999, Clin. Cancer Res. 5:1935-1944; E. M. Bailyes et al., 1997, Biochem. J. 327(Pt 1):209-215; see below).
Ninety percent of colorectal cancer tissue biopsies exhibit elevated IGF1R levels wherein the extent of IGF1R expression is correlated with the severity of the disease. Analysis of primary cervical cancer cell cultures and cervical cancer cell lines revealed 3- and 5-fold overexpression of IGF1R, respectively, as compared to normal ectocervical cells (Steller, et al., (1996) Cancer Res. 56:1762). Expression of IGF in synovial sarcoma cells also correlated with an aggressive phenotype (i.e., metastasis and high rate of proliferation; Xie, et al., (1999) Cancer Res. 59:3588).
Recent studies have also shown a connection between IGF-1 levels and ovarian cancer.
Potential strategies for inducing apoptosis or for inhibiting cell proliferation associated with increased IGF-I, increased IGF-II and/or increased IGF-IR receptor levels include suppressing IGF-I levels or IGF-II levels or preventing the binding of IGF-I to the IGF-IR. Anti-IGF-1R specific antibodies are contemplated to achieve this objective.
The association of expression levels of IGF-1R expressing cells with increased risk for one of breast, colon, pancreas, lung or ovarian cancer has been a consistent finding in a majority of epidemiologic studies. The progress in the understanding of cancer progression and early detection has been slow and frustrating due to the complex multifactorial nature and heterogeneity of the cancer syndrome. One of the challenges in drug development is to show in pre-clinical development, in clinical trials and with an approved agent that the anti-IGF-1R therapeutic is effective. One way to do this is to have suitable biomarkers that indicate when IGF-1R activity is inhibited. Currently employed diagnostic techniques such as medical imaging, tissue biopsy and bioanalytical assay of body fluids by enzyme linked immunosorbent assay (ELISA) are insufficiently sensitive and specific to detect most types of early-stage cancers. Moreover, these assays are labour intensive, time consuming, expensive and don't have multiplexing capability. To date, reliable diagnostic or prognostic IGF-1R specific markers have not been identified for any one or more of various IGF-1R mediated pathologies that could be effective in not only detecting tumors bearing IGF-1R expressing cells but also in monitoring treatment and gauging tumor aggressiveness. Indeed, the paucity of reliable biomarkers that show efficacy in detecting IGF-1R has hampered industry efforts in evaluating the efficacy of numerous anti-IGF-1R therapeutic protocols.
The present invention aims to provide at least one reagent that can be used as a diagnostic or prognostic biomarker for detecting and/or monitoring oncogenic disorders especially those characterized by expression of IGF-1R or those that are mediated by aberrant IGF0-1R expression.
Previous attempts to develop an antibody that can be used as a diagnostic or prognostic tool have not been reported. Described herein are novel antibodies that meet this criteria.
Other features and advantages of the invention will be apparent from the detailed description and examples that follow.